Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

Summary

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

Abstract

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

Introduction

The mission of a viral particle is to transport its genome from an infected host cell to a non-infected host cell and to deliver it into the cytoplasm or the nucleus in a replication-competent form¹. This process is initially triggered by binding to host cell receptors, followed by fusion of virion and cellular membranes. For enveloped viruses, like influenza viruses, the spike glycoproteins are responsible for receptor binding and fusion^1,2. Viral envelope glycoproteins (e.g., pyrogens, antigens), are involved in many important properties and events, such as virus lifecycle initiation (binding and ....

Protocol

1. Day 1: Cell Culture and Seeding

1. Cultivate human embryonic kidney (HEK) 293T/17 cells in 60 mm dishes with Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin (DMEM Complete Medium, DCM) in a 37 °C, 5% carbon dioxide (CO₂) incubator until about 80% confluent.
   NOTE: HEK 293T/17 low passage cells are recommended.
2. Carefully wash the cells with 5 mL of phosphate buffered saline (PBS) 1x.
   NOTE:.......

Discussion

In this protocol, we describe a method to produce influenza virus pseudotyped particles (pp) in a BSL-2 setting. The reporter plasmid pcDNA-GFP is incorporated into the pps and can be used to quantify pps by FACS in an infectivity assay. We chose two types of susceptible cell lines because they are widely used in influenza research. MDCK cells would provide a good control to the variable immortalized human cells used in these studies.

This protocol is based on the retrovirus MLV, which can inc.......

Materials

Name	Company	Catalog Number	Comments
Benzonase Nuclease	Millipore	70664	Effective viscosity reduction and removal of nucleic acids from protein solutions
Clear Flat Bottom Polystyrene TC-treated Microplates (96-well)	Corning	3599	Treated for optimal cell attachment Sterilized by gamma radiation and certified nonpyrogenic Individual alphanumeric codes for well identification
Clear TC-treated Multiple Well Plates (6-wells)	Costar	3516	Individual alphanumerical codes for well identification Treated for optimal cell attachment Sterilized by gamma irradiation
Dulbecco's modified essential medium (DMEM)	Gibco	11965092	A widely used basal medium for supporting the growth of many different mammalian cells
Fetal bovine serum	Excell	FND500	fetal bovine sera that can offer excellent value for basic cell culture, specialty research, and specific assays
Fluorescence Activated Cell Sorting (FACS)	Beckman coulter	cytoflex
Human alveolar adenocarcinoma A549 cells	ATCC	CRM-CCL-185
Human embryonic kidney (HEK) HEK-293T/17 cells	ATCC	CRL-11268	A versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines
Inverted fluorescent biological microscope	Olympus	BX51-32P01-FLB3
Inverted light microscope	Olympus	CKX31-12PHP
Lipofectamine 2000 Transfection Reagent	Invitrogen	11668019	Rapid, sensitive and precise probe-based qPCR detection and quantitation of target RNA targets.
Luna Universal Probe One-Step RT-qPCR Kit	NEB	E3006L	Will withstand up to 14,000 RCF RNase-/DNase-free Nonpyrogenic
Madin-Darby Canine Kidney (MDCK) cells	ATCC	CCL-34
MaxyClear Snaplock Microcentrifuge Tube (1.5 mL)	Axygen	MCT-150-C	33 mm, gamma sterilized
Millex-HV Syringe Filter Unit, 0.45 µm, PVDF	Millipore	SLHV033RS	an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in cell growth rate or morphology. Opti-MEM I medium is also recommended for use with cationic lipid transfection reagents, such as Lipofectamine reagent.
Opti-MEM I Reduced Serum Medium	Gibco	11058021	The antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective combined action against gram-positive and gram-negative bacteria.
penicillin-streptomycin	Gibco	15140122	Maximum RCF is 12,500 xg Temperature range from -80 °C to 120 °C RNase-/DNase-free Sterile
PP Centrifuge Tubes (15 mL)	Corning	430791	a stable and highly reactive serine protease
Proteinase K	Beyotime	ST532	Treated for optimal cell attachment Sterilized by gamma radiation and certified nonpyrogenic
TC-treated Culture Dish (60mm)	Corning	430166	Trypsin from bovine pancreas TPCK Treated, essentially salt-free, lyophilized powder, ≥10,000 BAEE units/mg protein
TPCK-trypsin	Sigma	T1426	This liquid formulation of trypsin contains EDTA and phenol red. Gibco Trypsin-EDTA is made from trypsin powder, an irradiated mixture of proteases derived from porcine pancreas. Due to its digestive strength, trypsin is widely used for cell dissociation, routine cell culture passaging, and primary tissue dissociation. The trypsin concentration required for dissociation varies with cell type and experimental requirements.
Trypsin-EDTA (0.25%), phenol red	Gibco	25200056